Tetrahydrobiopterin is a required cofactor for the hepatic phenylalanine hydroxylating system. De novo synthesis of tetrahydrobiopterin is regulated mainly by the activity of the first enzyme of the biosynthetic pathway, GTP cyclohydrolase I (GTPCH). The GTPCH feedback regulatory protein (GFRP) forms a complex with GTPCH and confers endproduct feedback sensitivity. GFRP mRNA distribution in rat brain has now been determined and correlated with GTPCH mRNA levels. All aminergic neurons contain both GFRP and GTPCH mRNA's, although the ratio of GFRP to GTPCH mRNA is much greater in serotonergic neurons than in dopaminergic neurons, suggesting different modes of regulation of tetrahydrobiopterin and neurotransmitter synthesis. The human gene for GFRP has been cloned and sequenced. It contains three exons and has been localized to chromosome 15q15. Tetrahydrobiopterin is also required by all forms of nitric oxide synthase and limits production of nitric oxide, endothelial derived relaxing factor, by endothelial cells and vascular smooth muscle cells. Availability of intracellular tetrahydrobiopterin likely plays a role in controlling vascular tone under normal and pathological conditions.